System for Glycated Protein Detection

ABSTRACT

A method of detecting the presence of glycated proteins or peptides (GPs) in a sample comprises carrying out the steps of assessing the sample for fluorescence, subjecting the sample to UV radiation, and reassessing the sample for an increase in fluorescence relative to any fluorescence assessed in said first assessing step, an increase in fluorescence at said reassessing step being indicative of the presence of GPs. The method may be useful for detecting disease such as diabetes.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/077,372, filed Jul. 1, 2008, the entirety of which is incorporatedherein by reference.

FIELD

The present invention relates to a system for the detection of glycatedproteins or peptides (GPs).

BACKGROUND

Glycation (sometimes called non-enzymatic glycosylation) is the resultof a sugar molecule, such as fructose or glucose, bonding to a proteinor lipid molecule without the controlling action of an enzyme. Glycationmay occur either inside the body (endogenous glycation) or outside thebody (exogenous glycation). Enzyme-controlled addition of sugars toprotein or lipid molecules is termed glycosylation; glycation is ahaphazard process that impairs the functioning of biomolecules, whereasglycosylation occurs at defined sites on the target molecule and isrequired in order for the molecule to function.

Exogenous glycations may also be referred to as dietary or pre-formed.Exogenous glycations and advanced glycation endproducts (AGES) aretypically formed when sugars are cooked with proteins or fats.Temperatures over 120° C. (˜248° F.) greatly accelerate the reactions,but lower temperatures with longer cooking times also promote theirformation.

These compounds are absorbed by the body during digestion with about 30%efficiency. Browning reactions are evidence of pre-formed glycations.Sugar is often added to products such as french fries and baked goods toenhance browning. Until recently, it was thought that exogenousglycations and AGEs were negligible contributors to inflammation anddisease states, but recent work has shown that they are important.Although most of the research work has been done with reference todiabetes, these results are most likely important for all people, asexogenous AGEs are implicated in the initiation of retinal dysfunction,cardiovascular diseases, type II diabetes, and many other age-relatedchronic diseases.

Food manufacturers have added AGEs to foods, especially in the last 50years, as flavor enhancers and colorants to improve appearance. Foodswith significant browning, caramelization, or with directly addedpreformed AGEs can be exceptionally high in these pro-inflammatory anddisease initiating compounds. A very partial listing of foods with veryhigh exogenous AGEs includes: donuts, barbecued meats, cake, and darkcolored fizzy drinks.

Endogenous glycations occur mainly in the bloodstream to a smallproportion of the absorbed simple sugars: glucose, fructose, andgalactose. The balance of the sugar molecules is used for metabolicprocesses. It appears that fructose and galactose have approximately tentimes the glycation activity of glucose, the primary body fuel.Glycation is the first step in the evolution of these molecules througha complex series of very slow reactions in the body known as Amadorireactions, Schiff base reactions, and Maillard reactions, all leading toadvanced glycation endproducts. Some AGEs are benign, but others aremore reactive than the sugars they are derived from, and are implicatedin many age-related chronic diseases such as: type II diabetes mellitus(beta cell damage), cardiovascular diseases (the endothelium,fibrinogen, and collagen are damaged), Alzheimer's disease (amyloidproteins are side-products of the reactions progressing to AGEs), cancer(acrylamide and other side-products are released), peripheral neuropathy(the myelin is attacked), and other sensory losses such as deafness (dueto demyelination) and blindness (mostly due to microvascular damage inthe retina). This range of diseases is the result of the very basiclevel at which glycations interfere with molecular and cellularfunctioning throughout the body and the release of highly-oxidizingside-products such as hydrogen peroxide.

Glycated substances are eliminated from the body slowly, since the renalclearance factor is only about 30%. This implies that the half-life of aglycation within the body is about double the average cell life. Redblood cells are the shortest-lived cells in the body (120 days), so thehalf-life is about 240 days. This fact is used in monitoring blood sugarcontrol in diabetes by monitoring the glycated hemoglobin level. As aconsequence, long-lived cells (such as nerves, brain cells),long-lasting proteins (such as eye crystalline and collagen), and DNAmay accumulate substantial damage over time. Metabolically-active cellssuch as the glomeruli in the kidneys, retina cells in the eyes, and betacells (insulin-producing) in the pancreas are also at high risk ofdamage. The epithelial cells of the blood vessels are damaged directlyby glycations, which are implicated in atherosclerosis, for example.Atherosclerotic plaque tends to accumulate at areas of high blood flow(such as the entrance to the coronary arteries) due to the increasedpresentation of sugar molecules, glycations and glycation end-productsat these points. Damage by glycation results in stiffening of thecollagen in the blood vessel walls, leading to high blood pressure.Glycations also cause weakening of the collagen in the blood vesselwalls, which may lead to micro- or macro-aneurisms; this may causestrokes if in the brain.

The USA, New Zealand and many developed nations are facing a dangerousepidemic of type 2 diabetes. In the US, there are an estimated 20.6million people with diabetes and 30% are undiagnosed. Another 54 millionpeople have some form of pre-diabetes and many will develop intodiabetes within three years.

Diagnosis of diabetes is typically initiated during a physicalexamination by a primary care physician. Screening for type 2 diabetesand pre-diabetes is inadequate. The most widely used test, the fastingplasma glucose (FPG) test, requires the subject to fast overnight beforebeing subjected to a blood draw. The sensitivity is poor (40-60%).Currently, around 50% of patients diagnosed with diabetes already haveone or more irreversible complications due to untreated diabetes.

International patent application WO 2005/045393 discloses a non-invasivemethod of determining a measure of glycation end-product or diseasestate using tissue fluorescence. A portion of the tissue of anindividual is illuminated with excitation light then light emitted bythe tissue due to fluorescence of certain chemicals (mainly AGEs) isdetected. The detected light can be combined with a model relatingfluorescence with a disease state to determine the disease state of anindividual. Various correction techniques are employed to reducedetermination errors due to detection of light other than that fromfluorescence of a chemical in the tissue. For example, backgroundfluorescence of the skin based on the individuals biological informationcan affect the measure of AGEs. This adds to the complexity of the testdevice.

This test device is very similar to a test device being developed byVeraLight in Albuquerque, N. Mex., USA. This uses five differentexcitation wavelengths of light between 350 nm and 450 nm and measuresthe spectrum of fluorescence from each of the five excitationwavelengths of light. The information is then analyzed with principalcomponent analysis, with the object of separating the fluorescence ofthe GPs from other fluorescence in the skin, and quantifying the amountof GPs.

OBJECT OF THE INVENTION

It is an object of the present invention to provide a system for thedetection of GPs which is an improvement on the above mentionedprocesses and/or which at least provides the public with a usefulchoice.

SUMMARY OF THE INVENTION

In one aspect of the invention there is provided a method of detectingthe presence of glycated proteins or peptides (GPs) in a samplecomprising carrying out the steps of assessing the sample forfluorescence, subjecting the sample to UV radiation, and reassessing thesample for an increase in fluorescence relative to any fluorescenceassessed in said first assessing step, the presence of fluorescence atsaid assessing step and an increase in fluorescence at said reassessingstep being indicative of the presence of GPs.

In some embodiments the physical state or phase of the sample is notaltered within the steps of the method.

In one form of the invention the sample is a solid material immobilisedon a support and the method includes subjecting the sample to UVradiation by irradiating the sample on the support with UV radiation andreassessment of the fluorescence of the sample.

In another alternative form the sample is a solution or a suspension,and the method includes subjecting the sample to UV radiation bysubjecting the solution or suspension to UV radiation.

In some embodiments the method includes measuring actual fluorescence ofthe sample. Additionally the method includes measuring any change inactual fluorescence between the assessment and the reassessment.

In some embodiments the fluorescence observed is spectrally resolved atthe assessment and reassessment steps. Additionally the shape of thefluorescence is analyzed for additional information that will give amore accurate indication of the presence of GPs.

In some embodiments the method comprises subjecting the sample betweenassessments to UV radiation in the wavelength range 200-320 nm, 240-320nm, 200-300 nm, or more preferably the UV radiation wavelength is in therange 250-260 nm or about 254 nm.

The exposure time for optimal enhancement depends upon the intensity ofthe UV radiation source and the level of GPs that may be present in thesample. The enhancement exposure time may be less than 20 minutes, lessthan 10 minutes, or less than 5 minutes.

In some embodiments the method includes assessing the fluorescence ofthe sample, subjecting the sample to a pulse of UV radiation andreassessing the fluorescence of the sample after a delay.

In some embodiments the delay is a period of time substantiallycorresponding to the fluorescence lifetime of a class of GPs.

In some embodiments the steps of assessing and reassessing thefluorescence include assessing and reassessing a broadband fluorescenceof the sample.

In some embodiments the steps of assessing and reassessing thefluorescence include causing the fluorescence to pass through a filteroriented to pass substantially only horizontally polarised light, andassessing and reassessing the fluorescence by reference to thehorizontally polarised fluorescent light. The excitation light may bevertically polarised. Alternatively the excitation light may beunpolarized light.

In some embodiments the sample may be subjected to a modulated UVsignal, and the fluorescence is reassessed for a modulated response. Thereassessment may be after a period of time substantially correspondingto the fluorescence lifetime of the GP fluorescence.

In another aspect of the invention there is provided a detector fordetecting GP in a sample comprising a UV source, a detection zone withinwhich the sample may be placed or may pass, means for fluorescenceanalysis arranged to assess for the presence of GPs without altering thestructure of any GP by assessing the sample for fluorescence, subjectingthe sample to UV radiation, and then reassessing the sample for anincrease in fluorescence relative to any fluorescence assessed in saidfirst assessing step, the presence of fluorescence at said assessingstep and an increase in fluorescence at said reassessing step beingindicative of the presence of GPs.

In some embodiments the output of the detector is actual fluorescencemeasurements of the two assessments.

Alternatively or additionally the output of the detector may comprise areading indicative of the change in fluorescence between the twoassessments.

In some embodiments the detector is arranged to assess and reassess thefluorescence of the sample by reference to substantially onlyhorizontally polarised fluorescence light. The excitation light may bevertically polarised. Alternatively the excitation light may behorizontally polarised.

In some embodiments the detector is arranged to subject the samplebetween assessments to UV radiation in the wavelength range 200-320 nm,240-320 nm, 200-300 nm, or more preferably the UV radiation wavelengthis in the range 250-260 nm or about 254 nm.

In some embodiments the detector is arranged to spectrally resolve thefluorescence observed at the assessment and reassessment steps.Additionally the detector may analyze the shape of the fluorescence foradditional information that may give a more accurate indication of thepresence of GPs.

In some embodiments the detector is arranged to assess and reassessbroadband fluorescence of the sample.

In some embodiments the detector is arranged to reassess thefluorescence of the sample after a delay of 0.1-10 ns and morepreferably the delay is a period of time that corresponds to thefluorescence lifetime of the GP fluorescence.

In some embodiments the sample may be subjected to a modulated UVsignal, and the fluorescence is reassessed for a modulated response.Preferably the reassessment is after a period of time substantiallycorresponding to the fluorescence lifetime of the GP fluorescence.

In another aspect of the invention there is provided a methodascertaining whether a sample being a portion of skin of an individual,due to its content of GPs, indicates the presence of a disease in theindividual comprising the steps of providing a detector which comprisesUV source, a detection zone within which a sample may be placed or maypass, means for fluorescence analysis arranged to assess for thepresence of GPs by reference to a first assessment of the sample forfluorescence, exposure of the sample to the UV source, and areassessment of the sample for an increase in fluorescence relative toany fluorescence assessed in said first assessing step, the presence offluorescence at said first assessment step and an increase influorescence at said reassessment step being indicative of the presenceof GPs, all without altering the structure of any GP; setting thesensitivity of the detector at a predetermined threshold above which adisease would be considered to be present, positioning the detector sothat the sample is in the detection zone, and reading or interpretingthe output of the detector as either

a. above the threshold and thus indicative of the presence of disease,or

b. below the threshold and thus not indicative of the presence ofdisease.

In some embodiments the physical state of the sample being a portion ofskin of an individual is not altered within the steps of the method.

In some embodiments the detector is adapted to detect and identifydiabetes.

In another aspect of the invention there is provided a method ofdetecting the presence of GPs in a sample comprising carrying out thesteps of assessing the sample for fluorescence, subjecting the sample toUV radiation, then reassessing the sample for fluorescence, andspectrally resolving fluorescence observed in the assessment andreassessment steps and analyzing the spectrally resolved fluorescencefor an increase in fluorescence indicative of the presence of GPs.

In a final aspect of the invention there is provided a detector fordetecting GPs in a sample comprising a UV source, a detection zonewithin which the sample may be placed or may pass, means forfluorescence analysis arranged to assess for the presence of GPs withoutaltering the structure of any GP by assessing the sample forfluorescence, subjecting the sample to UV radiation, then reassessingthe sample for fluorescence, spectrally resolving fluorescence observedin the assessment and reassessment steps, and analyzing the spectrallyresolved fluorescence for an increase in fluorescence indicative of thepresence of GPs.

To those skilled in the art to which the invention relates, many changesin construction and widely differing embodiments and applications of theinvention will suggest themselves without departing from the scope ofthe invention as defined in the appended claims. The disclosures and thedescriptions herein are purely illustrative and are not intended to bein any sense limiting.

DEFINITIONS

As used herein the following terms have the meanings given:

“fluorescence” means the emission of light of a longer wavelength by asource caused by exposure to light of a shorter wavelength from anexternal source.“fluorescence lifetime” refers to how long the fluorescence processexists after the sample is excited.“sample” means any sample of whatever form including particulate,powders such as for example milk or whey powder, on a surface static ormoving or airborne, in solution or suspension including cloudy liquidssuch as milk, and includes a portion of skin or a blood sample of aperson or animal or a derivative from blood, a sample such as a urine,lymph, faces, or hair sample, or a tissue sample including but notlimited to a biopsy of an artery or organ from a person or animal.“vertically polarised” and “horizontally polarised” in relation to lightare used with respect to the scattering plane or surface. In the casesof the samples under investigation, the surface may be the surface ofthe molecule, or a solid phase; it is relative to the direction of thelight and of the species which is responsible for light reflectionand/or absorption.“and/or” means “and” or “or”, or both.

As used herein “(s)” following a noun means the plural and/or singularforms of the noun.

The term “comprising” as used in this specification and claims means“consisting at least in part of”, that is to say when interpretingindependent paragraphs including that term, the features prefaced bythat term in each paragraph will need to be present but other featurescan also be present.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described by way of example only and withreference to the drawings in which:

FIG. 1: is a generalized flow diagram of the method of the invention.

FIG. 2: is a plot of the spectral response for the enhancement of GPfluorescence.

FIG. 3: is a schematic of an embodiment of a detector in accordance withthe invention.

FIG. 4: is a schematic of an alternative embodiment of a detector inaccordance with the invention.

FIG. 5: is a schematic of an alternative embodiment of a detector inaccordance with the invention.

FIG. 6: is plot of fluorescence intensity against emission wavelengthshowing the effect of using polarised light on the signal to noiseratio.

FIG. 7: is a plot of intensity against wavelength for the fluorescenceof a human forearm as discussed in Trial1 subsequently described.

FIG. 8: is a plot of intensity against wavelength for the fluorescencedifference before and after irradiation with UV radiation of a diabeticsubject as discussed in Trial 1.

FIG. 9: is a plot of intensity against wavelength for the fluorescencedifference spectrum of a subject where there is a family history ofdiabetes as discussed in Trial 1.

FIG. 10: is a plot of intensity against wavelength for the fluorescencedifference spectrum of a 22 year old female with no history of diabetesas discussed in Trial 1.

FIG. 11: is a plot of intensity against wavelength for the fluorescencedifference spectrum of a 23 year old male with no history of diabetes asdiscussed in Trial 1.

FIG. 12: is a scattergraph of fluorescence enhanced results fornon-diabetic and diabetic groups, referred to in the subsequentlydescribed Trial 2.

FIGS. 13 and 14: show an embodiment of a detector of the inventionincorporated in a desk.

FIG. 15: shows an embodiment of a portable desktop detector of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

We have found that GPs will exhibit fluorescence enhancement on UVexposure. The invention comprises:

-   -   assessing the fluorescence of a sample (such as but not limited        to a portion of skin of an individual) which is suspected of        containing GPs,    -   exposing the sample to ultraviolet radiation,    -   reassessing the fluorescence of the sample, and determining the        presence (or absence) of GPs.

If the fluorescence is increased after exposure to UV radiation thesample is assessed as containing GPs. The method is illustratedgenerally in the flow diagram of FIG. 1 (in which the wavelength rangesare given by way of example).

FIG. 2 shows the spectral response curve for the enhancement of the GPfluorescence. The full range of enhancement runs from about 230 nm to330 nm. However the peak enhancement occurs on exposure to UV light ofabout 254 nm. The squares show measured intensities. The line is a leastsquares fit of a Gaussian profile to the measured data. As the maximumenhancement is observed at or near 254 nm this is an ideal region of thespectrum for assessing fluorescence enhancement.

In the assessment and reassessment of fluorescence the sample may forexample be exposed to UV in the wavelength range 200-300 nm or 300-450nm and the fluorescence detected in the wavelength range 300-600 nm or300-450 nm. The fluorescence may be integrated over the wavelength rangeof each fluorescence assessment and the integrated results compared fora fluorescence increase, or increase beyond a difference threshold, asindicative of the presence of GPs.

Optionally the fluorescence information from the detector(s) may becorrected to reduce errors, due to detection of light other than fromfluorescence of glycated proteins or peptides for example. Thebackground fluorescence may be subtracted, and also optionally thefluorescence information may be adjusted to compensate for the colour ofthe skin of the subject by for example dividing all the values of thedetected spectrum by the values between 350-400 nm, to produce anormalized spectrum.

FIG. 3 schematically illustrates the functional elements one embodimentof a detector (and method) in accordance with the invention. The Figureschematically illustrates the sample 1, and detector elements being abroad band UV lamp 2 as the UV source, and a diode detector 3. The UVradiation is focussed onto the sample by means of a lens 6, and thelight passes through a long wavelength UV filter 4 (such as a 350 nmfilter). The fluorescence passes through a further filter 5 (such as a450 nm filter) to block the light that is simply reflected from thesample and to make certain that only the fluorescence will be detected,before being focused by means of a lens 7, and detected by the detector3. The amount of light detected for the purposes of this discussion willbe called measurement #1. The long wavelength UV filter 4 is then berotated out of the optical path and the sample is irradiated with allwavelengths of light from the lamp, including the short and midwavelength UV light. The detector is not used during this time. Finally,the long wavelength UV filter 4 is rotated back into the optical path.The fluorescence passes through filter 5 and lens 7 to detector 3. Ifthe intensity of the fluorescence at detector 3 has increased overmeasurement #1, or increased beyond a threshold margin over thefluorescence intensity of measurement #1, the sample may contain GPs.The amount of increase may be proportional to the amount of GPs.

FIG. 4 schematically illustrates the functional elements of anotherembodiment of a detector (and method) of the invention, that comprisesno moving parts (like the rotating filter used in FIG. 2). The Figureillustrates the sample 10, a UV lamp 12, which may be a UV lightemitting diode or a diode laser as the UV source, and a diode detector11. The UV radiation can pass through a long wavelength UV filter 13(such as a 350 nm filter) if the light from UV source is broadband. If aUV light emitting diode or UV diode laser is used the filter may not benecessary. The light might be further focussed on the sample with alens. The fluorescence can be collected by a second lens, and passesthrough a filter 14 (such as a 450 nm filter) to block light simplyreflected from the sample so that only the fluorescence is detected bythe detector 11. The sample is irradiated with a second UV lamp 15,emitting 250 to 300 nm UV light. This lamp may not need to be filtered,and can be focussed onto the sample by a lens. The detector 11 is notused during this time. Finally, the sample is again irradiated with theUV lamp 12 and the fluorescence is collected by a lens and passesthrough the filter 14 to detector 11. If the intensity of thefluorescence at detector 11 at the second measurement has increased overthe first, or increased beyond a threshold margin over the fluorescenceintensity of the first measurement, then the sample may contain GPs. Theamount of increase may be proportional to the amount of GPs.

FIG. 5 schematically illustrates a further embodiment of a detector (andmethod) of the invention in which the sample is moving, such as milkpowder moving on a conveyer belt. The Figure illustrates the sample 20,moving at a constant speed as shown by the arrow, a UV lamp 24, whichmay be a UV light emitting diode or a diode laser as the UV source, anda diode detector 21. The UV radiation can pass through a long wavelengthUV filter—not shown (such as a 350 nm filter) if the light from UVsource is broadband. If a UV light emitting diode or UV diode laser is aused a filter may not be necessary. The light may be further focussed onthe sample with a lens. The fluorescence can be collected by a secondlens (not shown), and passes through a filter 22 (such as a 450 nmfilter) to block light simply reflected from the sample, to detector 21.The sample is then irradiated by a second UV lamp 23, with 250 to 300 nmUV light. This lamp does not need to be filtered, but can be focussedonto the sample (no lens shown). Finally, the sample is again irradiatedwith the UV lamp 25, which might be a UV light emitting diode or a diodelaser as the UV source, and a diode detector 26. The UV radiation canpass through a long wavelength UV filter—not shown (such as a 350 nmfilter) if the light from UV source is broadband. If a UV light emittingdiode or UV diode laser is a used the filter will not be necessary. Thelight might be further focussed on the sample with a lens. Thefluorescence can be collected by a second lens (not shown), and passesthrough a filter 27 (such as a 450 nm filter) to block reflected light,to the detector 26. If the intensity of the fluorescence at detector 26has increased, or increased beyond a threshold margin over thefluorescence intensity of the first measurement, then the sample maycontain GPs. The measurements between detectors 21 and 26 must bedelayed by the time required for the sample to move between the twodetectors.

UV light sources include lamps (including fluorescent lamps, gas lamps,tungsten filament lamps, quartz lamps, halogen lamps, arc lamps, andpulsed discharge lamps, for example), and UV light emitting diodes,laser diodes, laser of any type capable of producing UV radiation (suchas gas, dye or solid state) and two-photon techniques where two separatephotons of differing wavelength as used to provide the requiredexcitation wavelength. For example, a 254 nm light to bring aboutfluorescence enhancement can be achieved from a high intensity of 508 nmlight. Two photons of 508 nm could be simultaneously absorbed to createthe same effect and response as one 254 nm photon being absorbed. Anadvantage of such a two-photon absorption is that all optics and thelight emitter work in the visible region of the spectrum, whilst theabsorption band of the sample is in the UV region. When subjecting thesample to UV radiation is referred to, scenarios such as this areincluded. It is the absorption band which should be considered in thiscase.

Other than with the two-photon method, the bandwidth of the excitinglight does not have to be very narrow. Thus some of the light sourcesdiscussed above may not need filtering, or could simply be filtered bygratings, interference fitters, coloured glass filters, or cut-offfilters.

The detector may be any photodetector for the detection of light,including, but not limited to, photodiodes, phototransisitors,photoresistors, photomultipliers, pyroelectric detectors, and chemicaldetectors, such as photographic plates. The detector can be a singleelement detector such as a photodiode that measures all light incidenton the detector window, or an image detector, such as a silver halideemulsion on a photographic plate or a CCD photodiode array. The detectorneeds to be sensitive to the range of wavelengths of light emitted fromthe fluorescing GPs.

A detection system of the invention may include means for analysis ofthe fluorescence, such as a computer processing apparatus which, forexample records fluorescence recorded or detected before irradiation andcompares it with that recorded or detected after, and identifies anyfluorescence enhancement indicating the presence of GPs. The analysismeans may determine actual fluorescence measurements or may simplydetermine the difference between the first and subsequent recording, anddetermine if an enhancement has been observed. The analysis means mayrecord and store the outputs or it may simply trigger a light forexample, if GPs (or GPs content greater than a threshold limit) aredetected.

Optionally polarised light may be used to improve the ratio of thefluorescence signal to the background signal. If the irradiating UVlight is polarised, although the scattered (background) radiation willpreserve the polarisation of this light, the fluorescence signal doesnot. Thus if the sample is irradiated with vertically polarised light,although the scattered light will remain vertically polarised, thatlight which is fluoresced from the sample (as a result of the presenceof GPs) is a mixture of horizontally polarised and vertically polarisedradiation. By measurement of only the horizontally polarised light,fluorescence is measured with little, if any, background scattering.Alternatively the excitation light may be unpolarized light, and onlythe horizontally polarised fluorescent light is measured.

The terms “vertical” and “horizontal” polarisation are used withreference to the “scattering surface”. In the preferred form of thisembodiment of the invention we use the 90 degree geometry betweenincident and scattered light, and the incident radiation is polarisedvertically (with respect to the scattering surface or phase). Thescattering surface or phase will depend upon the nature of the samplebeing investigated but is the molecule or species responsible forreflecting and/or absorbing the incident light. FIG. 6 is a plot ofintensity against wavelength showing fluorescence from GPs observed forthe arrangements of no polarisation; vertical-vertical polarisation(i.e. vertical incoming light; vertical detected fluorescence) andvertical—horizontal ((i.e. vertical incoming light; horizontal detectedfluorescence). This shows an improvement of signal to noise for thevertical-horizontal arrangement as discussed previously. This isparticularly true at the lower wavelengths.

Polarising filters may be incorporated into the previously describedembodiments, such as at least a horizontally polarising filter beforethe detector, and a vertically polarising filter may also be employedwith the UV source.

The wavelengths of fluorescence may be different depending upon thematerials present in the sample. Thus in a preferred embodiment, a morespecialised detector resolves the intensity of emission as a function ofwavelength, the shape of the fluorescence can be analyzed to determine amore accurate indication of the presence and amount of GPs in a sample.

Some embodiments of the invention may take advantage of the phenomenonthat fluorescence has a distinct lifetime. This lifetime is relative tothat of the scattered light, which has a zero lifetime. Specifically,after light is absorbed by the GP it takes a short amount of time forthe fluorescence to occur. This is usually between 0.1-10 ns. Thus ingeneral terms if following a short pulsed excitation, emitted lighthaving a zero lifetime is ignored and other emitted light detected, thecontribution to the emission by scattering is reduced and thus thesignal to noise ratio improved.

An alternative means of taking advantage of this phenomenon involvesmodulating the intensity of the light, for example in a sinusoidalfashion. The fluorescence of the GP follows the modulation of theexciting light, delayed by the fluorescence lifetime of the enhanced GP.Thus in this embodiment a modulated fluorescence signal is detected(again for example a sine wave type signal, if the exciting light wasmodulated accordingly) delayed by the fluorescence lifetime.

The invention provides a method for detection of GPs (which can becarried out non-invasively for human subjects). Thus the method may beimplemented, for example on a handheld detector.

The invention has importance in the detection of disease however thereare many other applications as would be known to one skilled in the art.

In the field of disease detection the invention may be useful fordetecting diabetes or a propensity towards diabetes in a living personor an animal and in particular type II diabetes, or other diseases suchas retinal dysfunction, or cardiovascular disease (including but notlimited to atherosclerosis, arteriosclerosis or arteriolosclerosis),obesity, metabolic syndrome, neurodegenerative disease, cancer, or renaldisease, for example. The method of the invention may be carried out insuch applications by non-invasively fluorescence—UVexposure-fluorescence reassessment of the skin of a living person, or ofa blood sample from the person. A detector arranged to carry out themethod of the invention may be implemented as a relatively smalldiagnostic instrument such as a table-top diagnostic instrument forexample, on a bed of which a person places, or into which a personinserts, his or her hand or forearm for fluorescence enhancementanalysis of the invention, or as a handheld diagnostic instrument, forexample. The presence of a GP in a sample from a person is indicative ofthe presence of a disease such as those listed above. In particular, thepresence of fluorescence at said assessing step and an increase influorescence at said reassessing step is indicative of the presence of aGP in a sample from a person, and therefore the presence of a disease inthe person.

FIGS. 13 and 14 show an embodiment of a detector of the inventionincorporated in a desk. A module 1 is mounted to the side of a desk 2.The top surface of the desk comprises one or more optical apertures 3from within the module 1. Within the module 1 are housed opticalcomponents of the detector such as one or more light sources such aslasers, one or more detectors, and electronics such as a computerprocessor and memory. Alternatively data from the detectors may besupplied to external computer processing apparatus such as a personalcomputer 5 connected to the module 1. In use a subject indicated at S inFIG. 14 places his or her forearm on the desktop over the opticalaperture(s) 3, before operation of the detector as described above asinitiated.

FIG. 15 shows an embodiment of a portable desktop detector of theinvention. The desk top instrument 10 comprises a hollow casing, mouldedfrom a plastics material for example, within which are housed opticalcomponents of the detector such as one or more light sources such aslasers, one or more detectors, electronics such as a computer processorand memory, and a power source typically a battery although the unit mayalso or alternatively comprise a power cord or socket for connection toan external power supply such as mains power or an external battery. Theinstrument is of a size that it can be conveniently carried by anindividual and includes a carry handle 11. The instrument may be placedon a table top or desktop for example, for use. An optical aperture 12is provided in the top surface of the casing through which the laser(s)and detector(s) of the instrument may operate. The top surface of theinstrument may be shaped as at 13 to position the forearm of a subjecton the instrument over the optical aperture 12 for use.

It is also worthy of note that it may be possible to detect single GPs.For example it is common to frequency double, triple and quadruple thelight from a Nd:YAG laser. With use of the tripled (355 nm) and thequadrupled (266 nm) light from a Nd:YAG laser, the resolution is suchthat single GPs may be detected by the method of the invention. Lasersother than the Nd:YAG could also be used, such as diode lasers.

The following description of trials work further illustrates theinvention.

Trial 1 Method

It is known that the level of glycated haemoglobin correlates very wellwith diabetes. A normal patient will have a level of glycatedhaemoglobin between 4.0 and 6.0%. A diabetic patient will have aglycated haemoglobin level above 6.9%. It is assumed that the level ofGPs in the skin (glycated collagen) will correlate with the levels ofglycated haemoglobin, and that measuring the amount of glycated collagenin skin is a screening tool for diabetes.

A portion of skin of each of the subjects referred to below was exposedto a single excitation wavelength of 350 nm, and emission wavelengthsfrom 380 to 600 nm were measured. Enhancement was generally performedwith a 5 W germicidal lamp emitting 254 nm wavelength light, next to theskin for 60 seconds, in some cases and 10 seconds in others. The skinportion was again exposed to the same excitation wavelength andfluorescence reassessed. FIG. 7 shows the typical fluorescence spectrumof skin, the excitation wavelength was 350 nm and the intensity wasmeasured in arbitrary units.

Results

FIG. 8 shows the difference spectrum (the spectrum after exposure to 254nm UV light for 60 seconds minus the spectrum before exposure to 254 nmlight) of a known type II diabetic subject. The intensity scale isarbitrary. The relative intensity is near 80,000 at the peak.

FIG. 9 shows the difference spectrum (the spectrum after exposure to 254nm UV light for 60 seconds minus the spectrum before exposure to 254 nmlight) of a 53 year old subject where both parents are diabetic. Theintensity scale is arbitrary. The relative intensity is near 50,000 atthe peak.

FIG. 10 shows the difference spectrum of a young 22 year old woman withno history of diabetes. The intensity of the peak is near 40,000. Thisis significantly lower than the peak of nearly 80,000 measured for thediabetic subject (FIG. 8).

FIG. 11 shows the difference spectrum of a 23 year old male with nohistory of diabetes. The spectrum appears very noisy. This is becausethe peak is only near 4000. This is significantly lower than the peak ofnearly 80,000 measured for the diabetic subject (FIG. 8).

Trial 2 Method

During a 10 week period clinical data and enhanced skin autofluorescence(ESAF) measurements were obtained from 33 live persons with diabetes and19 healthy, non-diabetic persons as controls. ESAF was measured inquadruplicate on the subjects' forearms and hands using acomputer-controlled fluorospectroscope. Initially, variable UV exposuretimes were used, and in the latter part of the study the UV exposuretime between fluorescence measurement and reassessment was 10 seconds.

Results

The mean ESAF results were approximately 30-fold higher in the diabeticgroup compared to the controls. FIG. 12 is a scatter graph comparing theESAF measurements in the diabetic and control groups, and shows thesignificantly higher readings in the patients with diabetes.

1. A method of detecting the presence of glycated proteins or peptidesin a sample comprising carrying out the steps of assessing the samplefor fluorescence, subjecting the sample to UV radiation, and reassessingthe sample for an increase in fluorescence relative to any fluorescenceassessed in said first assessing step, the presence of fluorescence atsaid assessing step and an increase in fluorescence at said reassessingstep being indicative of the presence of glycated proteins or peptides.2. A method according to claim 1 including subjecting the sample betweenassessments to UV radiation in the wavelength range 200-300 nm.
 3. Amethod according to claim 1 including subjecting the sample betweenassessments to UV radiation in the wavelength range 250-260 nm.
 4. Amethod according to claim 1 including subjecting the sample betweenassessments to UV radiation of about 254 nm wavelength.
 5. A methodaccording to claim 1 including subjecting the sample to UV radiationbetween assessments for less than 5 minutes.
 6. A method according toclaim 1 including measuring actual fluorescence of the sample in saidassessment steps.
 7. A method according to claim 6 including measuringany change in actual fluorescence between the assessment and thereassessment steps.
 8. A method according to claim 1 comprisingassessing and reassessing the fluorescence of the sample by reference tosubstantially only horizontally polarised fluorescent light.
 9. A methodaccording to claim 8 including causing the fluorescence to pass a filteroriented to pass substantially only horizontally polarised light.
 10. Amethod according to claim 9 including in the assessment and reassessmentsteps exposing the sample to vertically polarised light as theexcitation light.
 11. A method according to claim 1 including spectrallyresolving the fluorescence observed in the assessment and reassessmentsteps.
 12. A method according to claim 11 including spectrally resolvingthe fluorescence observed in the assessment and reassessment steps andanalyzing the shape of the fluorescence.
 13. A method according to claim1 including assessing the fluorescence of the sample, subjecting thesample to a pulse of UV radiation, and reassessing the fluorescence ofthe sample after a delay.
 14. A method according to claim 13 includingreassessing the fluorescence of the sample after a period of timesubstantially corresponding to the fluorescence lifetime of a class ofglycated proteins or peptides.
 15. A method according to claim 1including assessing the fluorescence of the sample, subjecting thesample to a modulated UV signal, and reassessing the fluorescence of thesample for a modulated response.
 16. A method according to claim 1wherein the sample is a portion of skin of a living person or animal ora sample from a living person or animal.
 17. A method according to claim1 wherein the sample is of or from a person and including assessing thesample for the presence of glycated proteins or peptides as indicativeof the presence of a disease in the person.
 18. A detector for detectingfor the presence of glycated proteins or peptides in a sample comprisinga UV source, a detection zone within which the sample may be placed ormay pass, means for fluorescence analysis arranged to assess for thepresence of glycated proteins or peptides by assessing the sample forfluorescence, subjecting the sample to UV radiation, and thenreassessing the sample for an increase in fluorescence relative to anyfluorescence assessed in said first assessing step, the presence offluorescence at said assessing step and an increase in fluorescence atsaid reassessing step being indicative of the presence of glycatedproteins or peptides.
 19. A detector according to claim 18 wherein saidsource or sources of UV radiation is or are operable to irradiate thesample with UV radiation having a wavelength in the range of 200-300 nmas the radiation having the wavelength effective to cause fluorescenceof the sample.
 20. A detector according to claim 18 wherein said sourceor sources of UV radiation is or are operable to irradiate the samplewith UV radiation having a wavelength of about 254 nm wavelength as theradiation having the wavelength effective to cause fluorescence of thesample.
 21. A detector according to claim 18 wherein said radiationdetector is arranged to detect substantially only horizontally polarizedfluorescent radiation.
 22. A detector according to claim 18 wherein saidsource or sources of UV radiation include a source of UV radiationoperable to irradiate the sample with vertically polarized UV radiationas the radiation having the wavelength effective to cause fluorescenceof the sample.
 23. A detector according to claim 22 wherein said sourceor sources of UV radiation include a source of UV radiation operable toirradiate the sample with vertically polarized UV radiation as theradiation having the wavelength effective to cause fluorescence of thesample.
 24. A detector according to claim 18 wherein said computerapparatus is arranged to spectrally resolve the first and secondflorescence signals.
 25. A detector according to claim 18 wherein saidcomputer apparatus is arranged to spectrally resolve the first andsecond fluorescence signals and analyse the shape of the fluorescencesignals.
 26. A detector according to claim 18 wherein said source orsources of UV radiation include a source of UV radiation operable toirradiate the sample with UV radiation having a wavelength effective tocause a photochemical change in the sample as a pulse of said UVradiation, and wherein said computer apparatus is arranged to recordsaid second fluorescence signal after a time period from the end of saidpulse of between 0.1-10 ns.
 27. A detector according to claim 18 whereinsaid source or sources of UV radiation is or are operable to irradiatethe sample with modulated UV radiation, and said radiation detector isarranged to detect a modulated fluorescence response.
 28. Apparatus fordetecting glycated proteins or peptides in a sample comprising: a sourceor sources of UV radiation disposed for irradiating a sample andoperable to irradiate the sample with UV radiation having a wavelengtheffective to cause fluorescence of the sample and subsequently with UVradiation having a wavelength effective to cause a photochemical changein the sample; a radiation detector disposed for receiving fluorescenceradiation from the sample when irradiated by said source or sources ofUV radiation and generating a fluorescence signal upon receiving saidfluorescence radiation; and a computer apparatus operatively connectedto said radiation detector or detectors and programmed to (a) record afirst fluorescence signal from said radiation detector after the sampleis first irradiated with UV radiation having a wavelength effective tocause fluorescence of the sample; (b) record a second fluorescencesignal from said radiation detector after the sample is irradiated withUV radiation having a wavelength effective to cause a photochemicalchange in the sample and then re-irradiated with UV radiation having awavelength effective to cause fluorescence of the sample; (c) comparesaid first and second fluorescence signals; and then (d) determine ifthere is any enhancement of the fluorescence of the sample to therebydetect the presence of glycated proteins or peptides.
 29. An apparatusaccording to claim 28 wherein said source or sources of UV radiationcomprise(s) first and second sources of UV radiation wherein the firstsource is operable to irradiate the sample with UV radiation having thewavelength effective to cause fluorescence of the sample and the secondsource is operable to irradiate the sample with UV radiation having thewavelength effective to cause a photochemical change in the sample, andsaid radiation detector comprises first and second radiation detectorswherein the first radiation detector is disposed for receiving thefluorescence radiation from the sample when irradiated by the firstsource UV radiation and generating the first fluorescence signal uponreceiving said fluorescence radiation and the second radiation detectoris disposed for receiving fluorescence radiation from the sample whenre-irradiated by the second source of UV radiation and generating thesecond fluorescence signal upon receiving said fluorescence radiation.30. A method of diagnosing diabetes in a person or a likelihood of aperson becoming diabetic, which comprises assessing the skin or a bloodsample of the person for fluorescence, subjecting the skin or bloodsample to UV radiation, and reassessing the skin or blood sample for anincrease in fluorescence relative to any fluorescence assessed in saidfirst assessing step, the presence of fluorescence at said assessingstep and an increase in fluorescence at said reassessing step beingindicative of the presence of diabetes or likelihood of the personbecoming diabetic.
 31. A method according to claim 17 wherein thedisease is selected from any one or more of retinal dysfunction,cardiovascular disease, obesity, metabolic syndrome, diabetes,neurodegenerative disease, cancer, and renal disease.
 32. A methodaccording to claim 31 wherein the disease is diabetes.